Assays for detecting modulators of cytoskeletal function

ABSTRACT

Described herein are methods of identifying compounds which modulate the activity of the cytoskeletal system. The methods are rapid, convenient and sensitive. Preferably, the method is used to identify lead compounds that can be used as therapeutics, diagnostics and agricultural agents. Generally, test compounds are added to two cytoskeletal components which bind to one another, to determine whether the binding is affected by the test compound. Wherein the binding is affected, a compound which modulates the cytoskeletal system is identified.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national entry of International ApplicationNo. PCT/US98/18368, filed on Sep. 3, 1998, which claims priority to U.S.Provisional Application No. 60/057,895, filed on Sep. 4, 1997 which isincorporated herein by reference in its entirety for all purposes. Thisapplication is also a Continuation-in-Part of U.S. application Ser. No.09/724,609, filed on Nov. 28, 2000, now U.S. Pat. No. 6,489,134, whichis a Divisional of U.S. application Ser. No. 09/226,772, filed Jan. 6,1999, now U.S. Pat. No. 6,207,403, which claims priority to U.S.Provisional Application No. 60/070,772, filed on Jan. 8, 1998.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported in part by Grant Numbers gm35252 gm38499,gm332289, gm40509, and gm46551 from the National Institutes of Health.The Government of the United States of America may have rights in thisinvention.

FIELD OF THE INVENTION

This invention relates to assays for identifying compounds that modulatethe activity of a cytoskeletal system (e.g. an actin/myosin system, atubulin system, etc.).

BACKGROUND OF THE INVENTION

The cytoskeleton constitutes a large family of proteins that areinvolved in many critical processes of biology, such as chromosome andcell division, cell motility and intracellular transport. Vale and Kreis(1993) Guidebook to the Cytoskeletal and Motor Proteins New York: OxfordUniversity Press; Alberts et al. (1994) Molecular Biology of the Cell,788-858). Cytoskeletal proteins are found in all cells and are involvedin the pathogenesis of a large range of clinical diseases. Thecytoskeleton includes a collection of polymer proteins, microtubules,actin, intermediate filaments, and septins, as well as a wide variety ofproteins that bind to these polymers (polymer-interacting proteins).Some of the polymer-interacting proteins are molecular motors (myosins,kinesins, dyneins) (Goldstein (1993) Ann. Rev. Genetics 27: 319-351;Mooseker and Cheney (1995) Ann. Rev. Cell Biol. 111: 633-675) that areessential for transporting material within cells (e.g., chromosomalmovement during metaphase), for muscle contraction, and for cellmigration. Other groups of proteins (e.g., vinculin, talin andalpha-actinin) link different filaments, connect the cytoskeleton to theplasma membrane, control the assembly and disassembly of thecytoskeletal polymers, and moderate the organization of the polymerswithin cells.

Given the central role of the cytoskeleton in cell division, cellmigration, inflammation, and fungal/parasitic life cycles, it is afertile system for drug discovery. Although much is known about themolecular and structural properties of cytoskeletal components,relatively little is known about how to efficiently manipulatecytoskeletal structure and function. Such manipulation requires thediscovery and development of specific compounds that can predictably andsafely alter cytoskeletal structure and function. However, at present,drug targets in the cytoskeleton have been relatively untapped. Previousstudies have been directed towards drugs that interact with thecytoskeletal polymers themselves (e.g., taxol and vincristine), andtowards motility assays. Turner et al. (1996) Anal. Biochem. 242 (1):20-5; Gittes et al. (1996) Biophys. J. 70 (1): 418-29; Shirakawa et al.(1995) J. Exp. Biol. 198: 1809-15; Winkelmann et al. (1995) Biophys. J.68: 2444-53; Winkelmann et al. (1995) Biophys. J. 68: 72S. In general,the studies on polymerization and motility were preliminary researchstudies performed in an effort to define the existing mechanisms ofthese actions. Although the cytoskeletal system has been characterizedto some extent, studies have not focused on the binding interactions ofthe polymers such as microtubules and actin with various polymer bindingproteins such as molecular motors with a specific goal of identifyingand characterizing modulators of such interactions that could havebiopharmaceutical and bioagricultural relevance. In particular, there isstill a need in the art to identify compounds which can be used tomanipulate the cytoskeletal system, particularly in regards to modifyingthe binding characteristics of the cytoskeletal components to oneanother. In particular, there is a need in the art to identify compoundsthat modulate the cytoskeletal binding interactions which can be used astherapeutics and/or diagnostics, as well as compounds which can be usedin the bioagriculture field (e.g. as pesticides). It is noted thatvirtually no effort has been directed to finding agents (e.g. drugs)that target cytoskeletal proteins that bind to the different filamentsand which are expected to provide targets of greater specificity andthereby provide fewer unwanted side effects when targeted with variousmodulators.

The invention herein provides convenient and rapid methods foridentifying compounds not previously known to modulate cytoskeletalfunction. In particular, this invention provides assay methods thatinclude methods for measuring binding interactions between cytoskeletalpolymers and cytoskeletal polymer-binding proteins that can be appliedto high-throughput screening to identify small molecules that modifythis interaction. The methods described herein include methods thatprovide high sensitivity and can be used in complex mixtures, including,but not limited to crude cell extracts.

SUMMARY OF THE INVENTION

The present invention provides a method of identifying andcharacterizing compounds which modulate the binding of two cytoskeletalcomponents. The methods are rapid, convenient and sensitive. Preferably,the method is used to identify lead compounds that can be used astherapeutics or diagnostics, or which can be used in the agriculturalfield.

In one embodiment, this invention provides high throughput assay(efficient screening of multiple samples) methods for testing multipletest agents (e.g., compositions and/or compounds) for their ability tomodulate cytoskeletal function. The methods generally involve adhering afirst cytoskeletal component to a solid support; contacting this firstcomponent with a second cytoskeletal component having an affinity forsaid first cytoskeletal component, in a (e.g. aqueous) reaction mixture;further contacting the reaction mixture with one or more, preferablymultiple test compositions to determine their ability to modulate thebinding affinity of the first and second cytoskeletal components; anddetecting changes in the binding affinity of the second cytoskeletalcomponent to the first cytoskeletal component at a test concentrationand a control concentration (e.g., zero concentration) of said testcompositions; wherein said detecting does not involve detecting theactive movement of the cytoskeletal components. Detection can be byoptical methods including, but not limited to total internal reflectionmicroscopy or confocal microscopy.

The assays of the invention can be adapted to a wide variety of solidsupport, including, but not limited to glass, plastic surfaces, metalsurfaces, or mineral (e.g., quartz or mica) surfaces. These assays areideal for very high throughput screening for drugs that alterinteractions between cytoskeletal polymers and their interactingproteins. Assays can be advantageously applied to a 96 well (or greater)plate format.

The invention particularly encompasses methods wherein the firstcytoskeletal component is indirectly adhered to the solid support, ifneed be in an oriented fashion (see below); wherein the firstcytoskeletal component is indirectly adhered to the solid support bybinding to an inactivated molecular motor which is bound to the support,wherein the first cytoskeletal component forms multiple arrays on asingle integrated support, wherein the second component is tagged with areporter molecule, particularly a fluorophore such as GFP. Wherefluorescence is detected, in a preferred embodiment the method ofdetection is total internal reflection microscopy. The assays can detectbinding in the presence of a cytoskeletal polymer, of a monomer of acytoskeletal polymer, or a molecular motor. In preferred embodiments,the signal to noise ratio is at least about 2-fold, more preferably atleast about 4-fold. The density of the first cytoskeletal component onthe solid support is at least 2 polymers/50μ², the concentration of thefirst cytoskeletal component is at least 10 ng/μ², and/or the throughputis at least about one sample/min.

In another embodiment this invention provides methods of identifying alead compound (e.g., therapeutic or bioagricultural) that modulatesactivity of a cytoskeletal system. In this embodiment, the methodspreferably involve providing an assay mixture comprising a firstcomponent of a microtubule system and a second component of acytoskeletal system, wherein said first component and said secondcomponent have an affinity for each other; contacting the assay mixturewith a test compound to be screened for the ability to modulate bindingbetween the first component and the second component; detecting adifference in the binding specificity or avidity of the first componentto the second component, at a test concentration and a controlconcentration of said compound to be screened, wherein said detectingdoes not involve detecting active movement of a component of saidcytoskeletal system, and wherein the difference in the bindingspecificity or avidity or affinity of the first component to the secondcomponent identifies a compound that modulates activity of acytoskeletal system.

In particularly preferred embodiments, the first and second componentsare selected from the group consisting of cytoskeletal polymers, motorproteins and cytoskeletal polymer binding proteins. Preferred first andsecond components can be components of an actin/myosin system, a tubulinsystem, or an intermediate filament system. Preferred first and secondcytoskeletal components include binding pairs selected from Table 1. Thereaction mixture can include a cell lysate. The methods can furtherinvolve entering the identity of a test compound that has a significanteffect on binding of the first component to the second component into adatabase of therapeutic or bioagricultural lead compounds. Inclusion inthe database can require that the test compound cause at least a 10%change in binding affinity or avidity between the first and secondcomponent. The assay can optionally additionally include contacting acell with a test compound whose identity is entered in said database;and detecting inhibition in the growth or proliferation of the cell. Inthe assays described herein, the first component can be labeled with alabel (e.g., a fluorescent label). Where the label produces an opticalsignal, detection is preferably by an optical method (e.g., microscopy).

The assay methods of this invention are well suited for high throughputscreening. Thus, in preferred embodiments, at least 50 test compoundsare screened simultaneously. Similarly, at least two different firstcomponent and second component pairs can be tested simultaneously. Thetest compounds can be members of a combinatorial library. In manyassays, the first or second cytoskeletal components or said secondcomponents are attached to a solid support.

In still another embodiment, this invention provides methods ofidentifying a therapeutic lead compound that modulates activity of acytoskeletal system, where the methods involve providing an assaymixture comprising a first component of a cytoskeletal system and asecond component of a cytoskeletal system, wherein the first and saidsecond components specifically bind to each other; contacting the assaymixture with a test compound to be screened for the ability to inhibitor enhance binding between the first and second component; and detectinga change in coupling between ATP hydrolysis and force generation;wherein said change indicates that said compound modulates activity of acytoskeletal system. In a particularly preferred embodiment, the firstand second components are not both tubulin or both actin or both tauprotein, however, one of the two components can be tubulin, actin or tauprotein;

In still another preferred embodiment, the method in accordance with thepresent invention comprises the step of providing at least one assaymixture comprising a first component of a cytoskeletal system and asecond component of a cytoskeletal system, wherein the first componentand the second component have an affinity for (e.g., specifically bindto) each other. This embodiment further comprises the step of contactingthe assay mixture with at least one test compound to be screened for theability to modulate binding between the first component and secondcomponent. Also included in this embodiment is the step of detecting adifference in the binding specificity or avidity of the first componentto the second component, at a test concentration and a controlconcentration of the compound to be screened. A difference in thebinding specificity or avidity indicates the presence of a compound thatmodulates activity of a cytoskeletal system.

In an embodiment in accordance with the present invention, the first andsecond components are selected from the group consisting of cytoskeletalpolymers such as microtubules, actin and intermediate filaments, motorproteins such as kinesin, dynein and myosin and polymer binding proteinssuch as Op18, tau protein or microtubule associated proteins (MAPs). Inone embodiment provided herein, wherein one of said cytoskeletalcomponents is a cytoskeletal polymer, the other cytoskeletal componentis not the same polymer. In another embodiment herein, wherein onecytoskeletal component is a tau protein, the other cytoskeletalcomponent is not the same tau protein.

The assay mixture described in accordance with the invention cancomprise a cell lysate. A single assay mixture or a plurality of assaymixtures can be provided. A single test compound can be provided to eachassay mixture, or more than one test compound can be provided to eachassay mixture. Wherein a plurality of assay mixtures are provided, oneor more assay mixtures can comprise a test compound which differs fromthe test compound of another assay mixture.

In accordance with the invention provided herein, one of the componentsof the cytoskeletal system can be adhered directly or indirectly to asolid support. Alternatively, the components can be in solution.

In one embodiment, at least one of the first or second cytoskeletalcomponents is labeled. The label can be selected from a wide variety ofreporter molecules. Reporter molecules include fluorophores, fluorescentproteins, and epitope tags.

The detection binding affinity, avidity, or specificity can beaccomplished by optical methods including, but not limited to platereaders and microscopy. Preferred microscopic methods include confocalor total internal reflection microscopy. In one embodiment, flowcytometry is used. Alternatively, the method of detection can be by amethod of detecting enzymatic activity, e.g., an ATPase assay.Alternatively, the method of detection can be by determining proteininteractions, i.e., a two hybrid system.

In a preferred embodiment, the difference in binding specificity,affinity or avidity detected is 10% or greater in either direction ofthat of the test concentration. In another embodiment, the differencefrom that of the test compound is or is greater than 20%, 40%, 60% or80% in either direction. In an alternative embodiment, the differencefrom that of the test compound is or is greater than 100%, 200%, 300%,500%, 800%, or 1000%. In one embodiment provided herein, the controlconcentration of the test compound is the absence of the test compoundto be screened.

The compounds to be screened can be selected from any number of origins.The test compounds include, but are not limited to, proteins, peptides,peptidomimetics, peptoids, saccharides, nucleic acids (DNA and RNA), andsmall organic molecules. The compounds can be from a library ofsynthetic or natural product sources and may be from a library createdusing combinatorial techniques. Preferably, the test compound is a smallmolecule. The small molecule is preferably 4 kilodaltons (kd) or less.In another embodiment, the compound is less than 3 kd, 2 kd or 1 kd. Inanother embodiment the compound is less than 800 daltons (D), 500 D, 300D or 200 D. In another embodiment wherein both of said components are insolution, a preferred method excludes single nucleotides as the compoundto be screened. Alternatively, this embodiment excludes molecules whichcan be hydrolyzed by one of the cytoskeletal components to providechemical energy. Another preferred embodiment excludes antibodies,particularly those previously known in the art to bind to cytoskeletalcomponents. Another embodiment excludes inositol phosphates.

Definitions

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to an element molecule orcomposition whose effect on the interaction between two or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a suitablecarrier.

The terms “isolated” “purified” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “fusion protein” refers to a protein (polypeptide) composed oftwo polypeptides that, while generally unjoined in their native state,are joined by their respective amino and carboxyl termini through apeptide linkage to form a single continuous polypeptide. It will beappreciated that the two polypeptide components can be directly joinedor joined through a peptide linker/spacer.

“Cytoskeletal function” refers to the biological roles of thecytoskeleton: to provide structural organization (e.g., microvilli,mitotic spindle) and to mediate motile events within the cell (e.g.,muscle contraction, mitotic contractile ring; pseudopodal movement,active cell surface deformations, vesicle formation and translocation).Cytoskeletal activity infers involvement in cytoskeletal function.Cytoskeletal activity includes the binding interaction of twocytoskeletal components.

“Molecular motor” is a cytoskeletal molecule that utilizes chemicalenergy to produce mechanical force, and drives the motile properties ofthe cytoskeleton.

“Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. This term includes cytoskeletal polymers and monomersthereof (e.g., actin filaments, microtubules, myosin filaments, 100 Å orintermediate filaments), molecular motors, and cytoskeleton associatedregulatory proteins (e.g., tropomyosin, alpha-actinin).

“Cytoskeletal polymer” refers to homo and heteropolymers that form partof the cellular cytoskeleton (e.g., actin filaments, microtubules,myosin filaments, etc.)

“Cytoskeletal system” refers to a collection of cytoskeletal componentsincluding monomers of cytoskeletal polymers that are typically foundassociated with each other in vivo.

“A monomer of a cytoskeletal polymer” refers to the monomeric subunit ofa cytoskeletal polymer, such as the alpha and beta tubulin subunits ofmicrotubules, the G-actin subunit of actin filaments, the monomer ofintermediate filaments, and the myosin subunits of myosin filaments.

An “actin/myosin system” refers to the collection cytoskeletalcomponents, including monomers, typically associated with actin and/ormyosin in vivo. Such components include, but are not limited to actin,myosin, actin binding proteins (e.g., ABP-50, ABP-120, ABP-280), actindepolymerizing factor (ADF), α-actinins, actobindin, actolinkin,annexins, caldesmons, calponin, capping proteins, cofilin, coronin,c-proteins, dematins, depactin, dystrophin, ezrin, fascin, fimbrin,gCap39, gelsolins, hisactophilin, insertin, MARCKS, myomesin andm-protein, nebulin, nuclear actin binding protein (NAB), paramyosin,ponticulin, profilins, proteins 4.1, radixin, sarcomeric m-creatinekinase, severin, small actin crosslinking proteins, spectrins, tenuin,thymosin β4 (Tβ4), titin, tropomodulin, tropomyosins, troponins, villin,vitamin D binding/Gc protein (DBP/Gc), 25 kDa inhibitor of actinpolymerization (25 kDa IAP) and 43 kDa protein (see, e.g., Kreis andVale (1995) Guidebook to the cytoskeletal and motor proteins. OxfordUniversity Press, Oxford, U.K.).

A “tubulin system” refers to a collection of cytoskeletal components,including monomers, typically associated with tubulin in vivo. Suchcomponents include, but are not limited to tubulin, chartins, MAP1A,MAP1B/MAP5, MAP2, MAP3, MAP4 (MAP-U), MARPS, pericentrin, radial spokeproteins, microtubule motors, STOPs, syncolin, tau, α/β tubulin,γ-tubulin, tubulin tyrosine ligase (TTL), tubulin carboxypeptidase(TCP), X-MAP, 205K MAP, and the like (see, e.g., Keris and Vale (1995)Guidebook to the cytoskeletal and motor proteins. Oxford UniversityPress, Oxford, U.K.).

An “intermediate filament system” refers to a collection of cytoskeletalcomponents, including monomers, typically associated with intermediatefilaments in vivo. Such components include, but are not limited tointermediate filaments, cytokeratins, desmin, epinemin, filaggrins,filensin, GFAP, α-internexin, lamins, nestin, neurofilament tripletproteins (e.g. NF-L, NF-M, NF-H), paranemin, peripherin, plectin,synemin, vimentin, and the like (see, e.g., Keris and Vale (1995)Guidebook to the cytoskeletal and motor proteins. Oxford UniversityPress, Oxford, U.K.).

“Solid support” means any solid surface, such as a bead or planar glass,a flexible or stiff membrane, a plastic, metal, or mineral (e.g., quartzor mica) surface, to which a molecule may be adhered. The solid supportcan be planar or have simple or complex shape. The surface to which themolecule is adhered can be an external surface or an internal surface ofthe solid support. Particularly where the surface is porous, themolecule is likely to be attached to an internal surface.

“Adhered to” or “attached” to a solid support denotes that one of saidfirst or second cytoskeletal components is directly or indirectly fixedto the solid substrate and that more than 95% of the first cytoskeletalcomponent remains associated with the solid support at least until allthe manipulations are completed and the level of binding is assessed.

“Adhered or attached in an oriented fashion” means that essentially allthe individual cytoskeletal components that bind to the solid support doso at some defined site or domain of the cytoskeletal component, suchthat a second site of the molecule (e.g., a catalytic domain) can freelyinteract with molecules.

“Spatially arranged to form distinct arrays” means that the cytoskeletalcomponent or components that adhere to the solid support are laid out inprecise patterns, such as rows of dots, or rows of squares, or lines.

“Modulate” means to increase or decrease (e.g. binding affinity, and/oravidity and/or specificity) relative to a control or test concentration.In one embodiment, the difference in binding specificity, or affinity,or avidity detected is at least 10%, or alternatively at least 20%, oralternatively at least 40%, or alternatively at least 60% oralternatively at least 80% in either direction (increased or decreased).In an alternative embodiment, the difference in affinity or avidity ofthe control from that of the test compound is or is greater than 100%,200%, 300%, 500%, 800%, or 1000%.

“Binding specificity” refers to the extent that a first molecule bindsto a second molecule in relation to whether the first molecule will alsobind to other (third, fourth and so on) molecules. The bindingspecificity can be determined by an in vitro binding assay according tostandard techniques known in the art. In a preferred embodiment, thephrase “binding specificity” or “specifically binds to” or whenreferring to a cytoskeletal component refers to a binding reaction whichis determinative of the presence of the protein or in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated assay conditions, a specified molecule (e.g. an antibodyspecific for a cytoskeletal component) binds to a particularcytoskeletal component and does not bind in a significant amount toother proteins present in the sample.

The phrase “having an affinity for” in the context of a firstcytoskeletal component having an affinity for a second cytoskeletalcomponent refers to the tendency of the first and second cytoskeletalcomponents to associate with each other in vivo. The association can bepermanent or transient and is typically characterized by a change in oneor more physical and/or chemical properties of one or both of thecomponents. In some preferred embodiments, the components that have anaffinity for each other specifically bind to each other.

“Binding avidity” as used herein is the strength of interactions betweenmultivalent components. Determinations of binding avidity are known inthe art as described at page 124 in Kuby (1992) Immunology, W.H. Freemanand Company, New York.

“Binding affinity” as used herein refers to the strength of the sum oftotal of noncovalent interactions between two molecules. Determinationsof binding affinity are known in the art as described at pages 122-124in Kuby (1992) Immunology, W.H. Freeman and Company, New York.

“Active movement” is movement that requires and utilizes chemical energy(as opposed to Brownian motion, passive dispersion, etc.).

“Reporter molecule” denotes a molecule that can be detected eithervisually (e.g., because it has color, or generates a colored product, oremits fluorescence) or by use of a detector that detects properties ofthe reporter molecule (e.g., radioactivity, magnetic field, etc.). Inone embodiment, reporter molecules allow for the detection of theinteraction of two molecules. Reporter molecules include labels(including ligands) which allows for detection, such as a radiolabel,fluorophores, chemiluminescence, biotin, streptavidin, digoxigenin,anti-digoxigenin, sugars, lectins, antigens, and enzyme conjugates. Thereporter molecule can be a protein that can be used as a direct orindirect label, i.e., green fluorescent protein (GFP), blue fluorescentprotein (BFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), luciferase, β-galactosidase, all commercially available, i.e.,Clontech, Inc. Moreover, one embodiment utilizes molecules that changetheir fluorescence or activity upon a change in binding.

In one embodiment, “detecting the binding” means assessing the amount ofa given second component that binds to a given first component in thepresence and absence of a test composition. This process generallyinvolves the ability to assess the amount of the second componentassociated with a known fixed amount of the first component at selectedintervals after contacting the first and second components. This may beaccomplished by attaching to the second component a molecule orfunctional group that can be visualized or measured (e.g., a fluorescentmoiety, a radioactive atom, a biotin that can be detected using labelledavidin) or by using ligands that specifically bind to the secondcomponent. The level of binding is detected quantitatively.

“A compound that binds to both the solid support and to the firstcytoskeletal component” refers to a compound that binds to the substrateessentially irreversibly, preferably through a covalent bond or througha multivalent attachment, and to the first cytoskeletal component withhigh affinity (an effective K_(D)=at least 10⁻⁸, preferably at least10⁻¹⁰, most preferably at least 10⁻¹²). “Effective K_(D)” refers tosituations wherein there are multiple attachment sites between twocytoskeletal components; the binding of multiple sites, each of whichhas lower affinity, provides for binding with an overall effectiveaffinity makes it seem like the binding affinity of the interactionbetween the two components is higher than it is.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the cytoskeletal system in vivo which can haveapplication in both human and animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders such as RheumatoidArthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic diseases such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma,Osler Webber syndrome, cardiovascular diseases such as hypertension,cardiac ischemia and systolic and diastolic dysfunction and fungaldiseases such as aspergillosis, candidiasis and topical fungal diseasessuch as tinea pedis.

A “diagnostic” as used herein is a compound that assists in theidentification and characterization of a health or disease state. Thediagnostic can be used in standard assays as is known in the art.

A “bioagricultural compound” as used herein refers to a chemical orbiological compound that has utility in agriculture and functions tofoster food or fiber crop protection or yield improvement. For example,one such compound may serve as a herbicide to selectively control weeds,as a fungicide to control the spreading of plant diseases, as aninsecticide to ward off and destroy insect and mite pests. In addition,one such compound may demonstrate utility in seed treatment to improvethe growth environment of a germinating seed, seedling or young plant asa plant regulator or activator.

The phrase “coupling between ATP hydrolysis and force generation” refersto the fact that many molecular motors are effective ATPases hydrolyzingATP to ADP to provide energy for force generation. The ATPase activityof the motor is often increased dramatically when the motor binds toanother cytoskeletal component such as a microtubule. An alteration inthe relationship between motor binding or force generation and ATPhydrolysis is a change in “coupling between ATP hydrolysis and forcegeneration.”

It is understood that the definitions which apply to the cytoskeletalsystem apply to embodiments which are drawn to specific components ofthe cytoskeletal system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows diagrams of different kinesin-GFP chimeras.

FIG. 2 illustrates binding interactions between fluorescent motors(green fluorescent protein fused to kinesin (K560-GFP), Ned (Ncd-GFP),or a motor chimera (NK-1-GFP)) and microtubule polymers. Bindinginteractions are shown in ATP (a low affinity state) and no nucleotide(a high affinity state). Imaging was performed using total internalreflection microscopy. Differences in fluorescent motor binding to thesurface-bound microtubule can be detected and quantitated byfluorescence intensity.

DETAILED DESCRIPTION

Provided herein are assays for the purpose of identifying compounds thatmodulate the cytoskeletal system. In preferred embodiments, the assayseffectively screen and identify agents that increase or decreaseinteractions that normally occur between components of the cytoskeletalsystem (e.g. actin/myosin interactions, etc.). It was a discovery ofthis invention that cytoskeletal component interactions, in oneparticularly preferred embodiment, motor/track and accessory proteininteractions provide novel targets for screening for compounds that areuseful as animal therapeutics, bioagricultural agents, and the like.Unlike assays for agents that target highly conserved molecules (e.g.,tubulin), in preferred embodiments. the assays of this inventionidentify agents that specifically target relatively variable molecules(e.g., motors and/or accessory proteins) and thus provide a means formodulating cytoskeletal activity with a specificity (e.g., species an/ortissue specificity) hitherto unknown. As is described in further detailbelow, the assays provide for the convenient use of a number ofdifferent cytoskeletal components and assay formats. Moreover, anycompound can be tested rapidly and efficiently.

I. Assays for Modulators of Cytoskeletal Component Interactions.

The assay methods of this invention generally involve either identifyingbinding interactions between one or more test agents and a component ofa cytoskeletal system, or, more preferably identifying the effect of oneor more test agents on the binding interactions between two (or more)components of a cytoskeletal system. The assays can be performed insolution or in solid phase, as individual assays or in highly parallel(e.g. high throughput) modalities, and with single or multiple testagents.

A variety of different assays for detecting compounds and compositionscapable of binding to a cytoskeletal component and of modulating thebinding of a second cytoskeletal component to a first cytoskeletalcomponent are used in the present invention. For a general descriptionof different formats for binding assays, including competitive bindingassays and direct binding assays, see Basic and Clinical Immunology, 7thEdition (D. Stites and A. Terr, ed.) (1991); Enzyme Immunoassay, E. T.Maggio, ed., CRC Press, Boca Raton, Fla. (1980); and “Practice andTheory of Enzyme Immunoassays,” in P. Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers, B.V.Amsterdam (1985).

A) Direct Binding Assays.

In direct binding assays the ability of one or more test compositions tobind to a cytoskeletal component (e.g. actin, myosin, kinesin, tubulin,etc.) including, but not limited to the components identified in Table1, is assayed. Simple binding assays are well known to those of skill inthe art. In one embodiment, either the test composition (agent) or thecytoskeletal component is labeled, the component and the agent arecontacted with each other and the association of the labeled moiety(component or agent) with the other binding partner (cytoskeletalcomponent where the test agent is labeled and test agent where thecytoskeletal component is labeled) is detected and/or quantified.Alternatively, both the cytoskeletal component and the test agent canboth be labeled and the association of the labels then indicatesbinding. The use of fluorescent labels capable of fluorescence resonanceenergy transfer (FRET) greatly facilitates the detection of such anassociation (e.g., the fluorescence of the labels is typically quenchedwhen they are brought into proximity to each other, see, e.g., Stryer(1978) Ann. Rev. Biochem., 47: 819-846.).

Direct binding assays can also be performed in solid phase where eitherthe test agent(s) or the cytoskeletal component is immobilized on asolid support. When the cytoskeletal component is immobilized it iscontacted with the test agent(s) (optionally labeled) and converselywhere test agent(s) are immobilized they are contacted with thecytoskeletal component(s) (optionally labeled). After washing awayunbound test agent and/or cytoskeletal component, remaining bound testagent/cytoskeletal component complexes indicate binding of the testagent(s) to the cytoskeletal component. Where the non-immobilized testagent or cytoskeletal component was labeled, detection of the labelassociated with the solid support provides a measure of testagent/cytoskeletal component binding. Fluorescence resonance energytransfer systems (FRET) are suitable for use in the solid phase as well.

Neither test agent nor cytoskeletal component need be labeled prior tothe assay. An “indirect” subsequently applied label (e.g. a labeledantibody specific for the cytoskeletal component or test agent(s)) canbe used to detect the test agent/cytoskeletal component in the testagent/cytoskeletal component complex.

It will be appreciated that neither test agent nor cytoskeletalcomponent need be labeled in the assays. Other means of detectingcomplex formation are known to those of skill in the art (e.g.electrophoresis, density gradient centrifugation, etc.).

B) Two-Component Inhibition Assays.

In “two-component inhibition assays”, the test agent(s) are assayed fortheir ability to alter the binding affinity, specificity or aviditybetween two (or more) components of a cytoskeletal system. In generalterms, one or both of the two components of a cytoskeletal system (or oftwo different cytoskeletal systems) are contacted with a test agent. Thebinding affinity or avidity of the two components is compared to thesame assay performed at a different (control) concentration of testagent. A difference in the binding affinity, specificity, or avidity ofthe two cytoskeletal components indicates that the test agent effectscytoskeletal function.

The “test” agent can be contacted with one or both of the components ofa cytoskeletal system before the two components contact each other, atthe same time, or after the two components have been allowed to contacteach other.

A wide variety of assays suitable for detecting the effect of testagent(s) on binding of two components of biological systems are wellknown to those of skill in the art. In preferred embodiments, suchassays are “competitive” in format where the test agent is screened forthe ability to compete with a second cytoskeletal component for specificbinding sites on a first cytoskeletal component and thereby alter thebinding between the two cytoskeletal components. However, it isrecognized that the test agent need not bind either of the cytoskeletalcomponents in order to effect changes (in the conformation or thechemical constitution of one or both of the cytoskeletal components andthereby alter their binding interaction. Regardless of the specificmechanism of action of the test agent, the assays described below aregenerally designed to reveal alterations in the binding specificity,affinity, or avidity of the cytoskeletal components.

In one preferred embodiment, the test agent(s) are assayed relative to acontrol assay. The control assay can contain the test agent(s) at adifferent concentration or one or more particular test agent(s) can beabsent from the control assay. A difference (preferably a statisticallysignificant difference) in the cytoskeletal component binding betweenthe test and the control assays indicates that the test agent has aneffect on cytoskeletal function. An increase or decrease in binding byat least 10%, more preferably by at least 20%, most preferably by atlease 50%, 80%, 90% or more is preferred to record a positive result(indicating test agent activity) in an assay.

In yet another embodiment, one of the cytoskeletal components has adetectable moiety attached thereto, i.e., fluorescence, which changes inintensity, spectrum or polarization upon different binding thereto,i.e., the fluorescence changes upon a second agent binding thereto. Inyet another embodiment, a conjugate such as a phosphatase is used suchthat binding is measured upon adding substrate on which the enzyme canact. Moreover, it is understood that magnetic beads and the like can beused.

In an alternative embodiment, the binding affinity between a firstcytoskeletal component and a second cytoskeletal component is determinedin the presence and absence of a test agent. A difference in the bindingaffinity indicates the identification of a compound which modulates thecytoskeletal system. The invention also provides for the identificationof a compound which modulates the binding of a cytoskeletal component toanother cytoskeletal component.

1) “Competitive” Assays.

In “competitive” assays the test agent is assayed by contacting theagent with either or both of two components of a cytoskeletal system (afirst component e.g. a motor, and a second component, e.g. a “track”)that typically associate together. The effect of the test agent on thecytoskeletal system is then assayed by assessing the amount of secondcytoskeletal component associated with the first cytoskeletal component.Where the test agent actually competes with the for binding to one ormore of the cytoskeletal components or otherwise inhibits binding, theamount of a second cytoskeletal component associated with the firstcytoskeletal component is diminished relative to a control assay havinga lower concentration of the particular test agent or lacking that testagent at all.

Conversely, “agonistic” test agents may increase the binding affinity,avidity, or specificity of the two cytoskeletal components. In thisinstance, an increase the amount of a second cytoskeletal componentassociated with the first cytoskeletal component is increased relativeto a control assay having a lower concentration of the particular testagent or lacking that test agent at all.

The amount of inhibition or stimulation of binding of a cytoskeletalcomponent by the test compound depends on the binding assay conditionsand on the concentrations of cytoskeletal components, and test agent(s)used. Under specified assay conditions, a test agent is said to becapable of inhibiting or enhancing the binding of a second cytoskeletalcomponent to a first cytoskeletal component if the amount of boundsecond cytoskeletal component is decreased or increased, respectively,by a statistically significant amount. An increase or decrease inbinding by at least 10%, more preferably by at least 20%, mostpreferably by at lease 50%, 80%, 90% or more is preferred to record apositive result (indicating test agent activity) in an assay.

As indicated above, those skilled in the art understand that to affectthe binding between two cytoskeletal components, the test component neednot compete with the cytoskeletal components for a specific bindingsite. Therefore, in an embodiment herein, the modulator may induce achange in the conformation of the binding site so as to increase ordecrease binding.

As indicated above, the assays can be performed in solution or in solidphase. In a preferred solid phase assay, one of the cytoskeletalcomponents is attached to a solid support. The second cytoskeletalcomponent is then contacted with the first cytoskeletal component eitherprior to or after one or both components are exposed (contacted with)the test agent(s). After an appropriate wash step, the amount ofcytoskeletal component bound is assayed, e.g., as described below.

The amount of binding of the second cytoskeletal component to the firstcytoskeletal component can be assessed by directly labeling the secondcomponent with a detectable moiety, or by detecting the binding of alabeled ligand that specifically binds to the second cytoskeletalcomponent. A wide variety of labels may be used. The component may belabeled by any one of several methods. Traditionally, a radioactivelabel (³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P) is used. Non-radioactive labels includefluorophores, chemiluminescent agents, enzymes, and antibodies. Thechoice of label depends on sensitivity required, ease of conjugationwith the compound, stability requirements, and availableinstrumentation. A preferred fluorophore is a fluorescent protein (e.g.GFP). For a review of various labeling or signal producing systems whichmay be used, see U.S. Pat. No. 4,391,904.

2) FRET Assays.

As indicated above, fluorescent resonance energy transfer (FRET) systemscan also be used to assay protein protein interactions. In FRET-basedassays, both components (e.g. both cytoskeletal components) are labeledwith fluorescent labels. The absorption and emission spectra of thelabels are selected such that one label emits at a wavelength that theother absorbs. When the labels are brought into proximity to each other(e.g., by binding of the two cytoskeletal components to each other) theyquench thereby decreasing the fluorescence of the mixture. FRET is apowerful technique for measuring protein-protein associations and hasbeen used previously to measure the polymerization of monomeric actininto a polymer (Taylor et al. (1981) J. Cell Biol., 89: 362-367) andactin filament disassembly by severing (Yamamoto et al. (1982) J. CellBiol., 95: 711-719), but has not been used to screen for agents thatmodulate cytoskeletal component interactions.

Other assays can detect changes in fluorescence polarization.

3) Liquid Crystal Assay Systems.

In still another embodiment, binding of the two components of thecytoskeletal system can be detected by the use of liquid crystals.Liquid crystals have been used to amplify and transducereceptor-mediated binding of proteins at surfaces into optical outputs.Spontaneously organized surfaces can be designed so that a firstcytoskeletal component, upon binding to a second cytoskeletal component(e.g. microtubules) hosted on these surfaces, trigger changes in theorientations of 1- to 20-micrometer-thick films of supported liquidcrystals, thus corresponding to a reorientation of ˜10⁵ to 10⁶ mesogensper protein. Binding-induced changes in the intensity of lighttransmitted through the liquid crystal are easily seen with the nakedeye and can be further amplified by using surfaces designed so thatprotein-ligand recognition causes twisted nematic liquid crystals tountwist (see, e.g., Gupta et al. (1998) Science, 279: 2077-2080). Thisapproach to the detection of ligand-receptor binding does not requirelabeling of the analyte, does not require the use of electroanalyticalapparatus, provides a spatial resolution of micrometers, and issufficiently simple that it is useful in biochemical assays and imagingof spatially resolved chemical libraries.

4) ATPase Assay

In an alternative embodiment, used to identify agents that modulatebinding of cytoskeletal components, an ATPase assay can be used. Forexample, molecular motors are effective ATPases hydrolyzing ATP to ADPto provide energy for force generation. The ATPase activity of the motoris often increased dramatically when the motor binds to anothercytoskeletal component such as a microtubule. By examining ATPhydrolysis from a molecular motor in the presence of varyingconcentrations of test compounds which may effect binding between twocytoskeletal components, the binding can be quantified, therebyidentifying novel agents which modulate binding. This assay has not beendone to identify binding modulators.

One such ATPase assay is described in the examples below. In onepreferred embodiment, the ATPase activity assay utilizes 0.3 M PCA(perchloric acid) and malachite green reagent (8.27 mM sodium molybdateII, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-100). Toperform the assay, 10 μL of reaction is quenched in 90 μl of cold 0.3 MPCA. Phosphate standards are used so data can be converted to mMinorganic phosphate released.

When all reactions and standards have been quenched in PCA, 100 μl ofmalachite green reagent is added to the to relevant wells in e.g., amicrotiter plate. The mixture is developed for 10-15 minutes and theplate is read at an absorbance of 650 nm. If phosphate standards wereused, absorbance readings can be converted to mM Pi and plotted overtime.

5) Protein Protein Interaction (e.g. Two-Hybrid) Assay.

In yet another embodiment, protein-protein interactions are used. Forexample, a two-hybrid system as known in the art can be used. Thetwo-hybrid system is a method used to identify and clone genes forproteins that interact with a protein of interest. Briefly, the systemindicates protein-protein interaction by the reconstitution of GAL4function, which is detectable and only occurs when the proteinsinteract. This system and general methodologies concerning thetransformation of yeast with expressible vectors are described inCheng-Ting et al. (1991) Proc. Natl. Acad. Sci. USA, 88:9578-9582,Fields and Song (1989) Nature, 340:245-246, and Chevray and Nathans,(1992) Proc. Natl. Acad. Sci. USA, 89: 5789-5793.

C) Assaying Multiple Agents.

While each assay mixture can be utilized to assay the effect of a singletest agent, it will be recognizes that multiple test agents can also bescreened in a single assay mixture. In such a multi-agent assayembodiment, two or more, preferably 4 or more, more preferably 16 ormore and most preferably 32, 64, 128, 256, or even 512 or more agentsare screened in a single assay reaction mixture. A positive result inthat assay indicates that one or more of the combined agents aremodulators of cytoskeletal function. In this instance, in a preferredembodiment, the method is repeated wherein the candidate agents areseparated out to identify the modulator individually, or to verify thatthe agents work in conjunction to provide the difference in bindingspecificity, affinity, or avidity. Thus, for example, an assayoriginally run with 16 test agents may be re-run as four assays eachcontaining four of the original 16 test agents. Again those assaystesting positive can be divided and re-run until the agent or agent(s)responsible of the positive assay result are identified.

It is also noted that multiple test agents can be assayed together toidentify agents that are additive or even synergistic in their effect ona cytoskeletal system, or conversely, to identify test agent(s) that areantagonistic in their effects on a cytoskeletal system.

In one embodiment, the method further comprises the step of entering theidentity of a test compound which has been identified to modulateactivity of a cytoskeletal system in accordance with the presentinvention into a database of therapeutic, diagnostic or bioagriculturallead compounds. In some cases it may be desirable to perform furtherassays on the compounds which have been identified herein. For example,activity of the identified compounds can be further assessed in areasother than their ability to modulate binding. For example, their abilityto affect growth or proliferation of cells, particularly tumor cells,vesicle migration, mitosis, congression, filament movement, motility,etc. can be assessed.

D) High Throughput Screening.

In one embodiment, the assays of the present invention offer theadvantage that many samples can be processed in a short period of time.For example, plates having 96 or as many wells as are commerciallyavailable can be used. In addition, the cytoskeletal components can beattached to solid supports and spatially arranged to form distinctarrays, such as rows of dots or squares, or lines. This, coupled tosophisticated masking, assay and readout machines greatly increase theefficiency of performing each assay and detecting and quantifying theresults. It is possible with current technologies to efficiently makevast numbers (10⁶ or more) of peptides having specified sequences andarray them at distinct locations in a chip, and then to detectfluorescent associated with each position of the chip. See, e.g., U.S.Pat. No. 5,143,854; PCT Publication Nos. WO 90/15070, WO 92/10092 and WO93/09668; and Fodor et al. (1991) Science, 251, 767-77.

Conventionally, new chemical entities with useful properties (e.g.,inhibition of myosin tail interactions) are generated by identifying achemical compound (called a “lead compound”) with some desirableproperty or activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds.However, the current trend is to shorten the time scale for all aspectsof drug discovery. Because of the ability to test large numbers quicklyand efficiently, high throughput screening (HTS) methods are replacingconventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of compounds (testcompounds) potentially having the desired activity. Such “combinatorialchemical libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity(e.g., therapeutic or bioagricultural). The compounds thus identifiedcan serve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics or bioagricultural agents.

Any of the assays for the test compounds and compositions describedherein are amenable to high throughput screening. These assays detectinhibition of the characteristic activity of the cytoskeletal component,or inhibition of or binding to a receptor or other transduction moleculethat interacts with the cytoskeletal component.

High throughput assays for the presence, absence, or quantification ofparticular nucleic acids or protein products are well known to those ofskill in the art. Binding assays are similarly well known. Thus, forexample, U.S. Pat. No. 5,559,410 discloses high throughput screeningmethods for proteins, U.S. Pat. No. 5,585,639 discloses high throughputscreening methods for nucleic acid binding (i.e., in arrays), while U.S.Pat. Nos. 5,576,220 and 5,541,061 disclose methods of screening forligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc., Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.) These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols for the various high throughputassays. Thus, for example, Zymark Corp. provides technical bulletinsdescribing screening systems for detecting the modulation of genetranscription, ligand binding, and the like.

In a preferred embodiment of this invention, the signal to noise ratiois relatively high (the signal is at least about 4-fold, preferablyabout 10-fold, and more preferably about 100 fold above background).Where the first cytoskeletal component on the solid support is a polymer(e.g., F-actin or microtubule), the density of is at least 2, preferablyat least 10, more preferably at least 100, most preferably at least 1000polymers/50μ², wherein the average length of the polymers is 1μ. Theconcentration of the cytoskeletal component is at least 10, preferablyat least 100, and more preferably at least 1000 ng/μ². The desired highthroughput rate averages at least about one, preferably at least about10, and more preferably at least about 100 different test agents/min.

II. Assay Components.

A) Cytoskeletal Components

The number and identity of cytoskeleton components that have beenidentified thus far are legion, and far too numerous to be completelylisted here. A partial listing can be found in the following references:Vale and Kreis (1993) Guidebook to the Cytoskeletal and Motor ProteinsNew York: Oxford University Press; Goldstein (1993), Ann. Rev. Genetics27: 319-351; Mooseker and Cheney (1995) Annu. Rev. Cell Biol. 11:633-675; Burridge et al. (1996), Ann. Rev. Cell Dev. Biol. 12: 463-519.Of special interest are components associated with the actin filamentsystem (e.g., actin, myosin, tropomyosin, α-actinin, thymosin, profilin,spectrin, ankyrin, fimbrin, filamin, vinculin, villin, gelsolin,severin), with the microtubule system (alpha and beta tubulin, dynein,kinesin, MAPS, tau), and with the intermediate filaments (keratins,vimentin, neurofilament proteins, lamins, desmin). Although the assaysof the invention will often focus on interactions between cytoskeletalcomponents from the same filament subsystem (e.g., actin andactin-binding proteins), it should be noted that the different filamentsystems are integrated and that some components bridge more than onesystem and therefor components from two different systems can utilizedin an assay of this invention.

In a preferred embodiment herein, the first and second components areselected from the pairs shown in Table 1.

TABLE 1 Pairwise combinations of cytoskeletal interactions. ActinDepolymerizing Factor/Cofilin Actin Adducin Actin Alpha actinins ActinAlpha catenin Actin Annexins Actin Adenomatous Polyposis Coli ProteinTubulin/Microtubules Arp ⅔ Actin Axonemal Dynein Tubulin/MicrotubulesBipolar Kinesin Tubulin/Microtubules BPAG1 Intermediate FilamentsCaldesmon Actin Capping Protein Actin Cardiac Muscle Myosin Actin CENP-EBub1 CENP-E CENP-F Centrosomin Tubulin/Microtubules ChromokinesinTubulin/Microtubules CLIP-170 Tubulin/Microtubules Coronin ActinCortexillins Actin C-terminal Motor Domain Kinesin Tubulin/MicrotubulesCytoplasmic Dynein Tubulin/Microtubules Cytoplasmic Myosin II ActinDynactin Complex Dynein Dystrophin/Utrophin Actin ERM proteins: Ezrin,Radixin and Actin Moesin Filaggrins Intermediate Filaments Gamma TubulinTubulin/Microtubules Gelsolins Actin Heterotrimeric KinesinTubulin/Microtubules IFAP 300 Intermediate Filaments Internal MotorDomain Kinesin Tubulin/Microtubules Katanin Tubulin/Microtubules MAP1ATubulin/Microtubules MAP1B-MAP5 Tubulin/Microtubules MAP2Tubulin/Microtubules MAP4 Tubulin/Microtubules MARCKS Actin MARK ProteinKinases Tubulin/Microtubules Mitotic Kinesin Tubulin/MicrotubulesMonomeric Kinesin Tubulin/Microtubules Myosin Heavy Chain Kinases MyosinMyosin I Actin Myosin IX Actin Myosin Light Chain Kinases Myosin LightChains Myosin V Actin Myosin VII Actin NuMa Tubulin/MicrotubulesOp18/Stathmin Tubulin/Microtubules Pericentrin Tubulin/MicrotubulesPlectin Intermediate Filaments Profilin Actin Protein 4.1 Actin SeverinActin Smooth Muscle Myosin Actin Spectrins Actin STOPSTubulin/Microtubules Syncolin Tubulin/Microtubules Talin Actin TauProtein Tubulin/Microtubules Tensin Actin Thymosin beta 4 ActinTropomodulin Actin Tropomyosin Actin Troponins Actin VASP Actin VillinActin Vimentin Intermediate Filaments Vinculin Actin WASP ActinXMAP215/TOG Tubulin/Microtubules ZW10 Dynamitin ZW10 Rough DealActophorin Actin Zyxin Actin Merlin Actin Desmoplakin Vimentin ZonulaOccludin-1 Actin/spectrin Depactin Actin Ankyrin IntermediateFilaments/spectrin DNase Actin Filamin/Plastin Actin Fimbrin Actin ActAActin KIF Tubulin/Microtubules ABP-120 Actin EB1 Tubulin/MicrotubulesKIFs Tubulin/Microtubules Cdc42 Actin

In an alternative embodiment provided herein, the first componentdiffers from the second component. By “differs”, this includesembodiments wherein the components are the same but for a chemicalmodification of one but not the other. Moreover, if two componentsbelong to the same class of cytoskeletal components, i.e., are bothkinesins, but are distinct from one another based on their amino acidsequence, they are considered to differ from one another.

The cytoskeletal components of this invention can be recombinantlyexpressed using standard methods well known to those of skill in theart. Alternatively, the cytoskeletal components can obtained bypurification from natural sources as explained below.

In general, the cytoskeletal components of this invention may bepurified to substantial purity from natural and recombinant sources byknown protocols using standard techniques (Vale and Kreis (1993),Guidebook to the Cytoskeletal and Motor Proteins New York: OxfordUniversity Press), including differential extraction, selectiveprecipitation with such substances as ammonium sulfate, columnchromatography, immunopurification methods, and others. See, forinstance, Scopes (1982) Protein Purification: Principles and Practice,Springer-Verlag: New York. For example, cytoskeletal proteins andpolypeptides produced by recombinant DNA technology may be purified by acombination of cell lysis (e.g., sonication) and affinity chromatographyor immunoprecipitation with a specific antibody to cytoskeletalcomponents. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredpolypeptide. The proteins may then be further purified by standardprotein chemistry techniques.

The assays of the present invention also support the use of unpurifiedcytoskeletal components (e.g., cell lysates). Wherein a cell lysate ispresent, the lysate can be from any cell type (e.g., prokaryotic,eukaryotic, vertebrate, invertebrate and mammalian, etc.) Whereunpurified preparations are used, detection of cytoskeletal componentinteraction preferably involves the use of detection systems specificfor at least one of the cytoskeletal components studied.

B) Labeling of Cytoskeletal Components and/or Test Agent(s).

In one embodiment, the cytoskeletal component and/or the test agent(s)are labeled. Detectable labels suitable for use in the assays of thisinvention include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others such as thosecommonly used in an ELISA), and colorimetric labels such as colloidalgold or colored glass or plastic (e.g. polystyrene, polypropylene,latex, etc.) beads. Patents teaching the use of such labels include U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, ease of conjugation of thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc., and fluorescent proteins (e.g., GFP). Chemiluminescent compoundsinclude, but are note limited to, luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see, U.S. Pat.No. 4,391,904.

C) Solid Supports

As indicated above in one embodiment, the assays of this invention areperformed in the solid phase (e.g. with a test agent or a cytoskeletalcomponent attached to a solid support). The “solid support” can be madeof any material to which a molecule may be adhered that is compatiblewith the conditions and solutions for performing the binding assays.Examples include beaded or planar glass, metals, plastics, or minerals(e.g., mica or quartz). The supports can be relatively rigid, formed asdeformable sheets or membranes, or manufactured into useful assaydevices (e.g., microtiter dish (e.g., PVC, polypropylene, orpolystyrene), a test tube (glass or plastic), a dipstick (e.g. glass,PVC, polypropylene, polystyrene, latex, and the like), a microcentrifugetube) and the like. A preferred support is aminosilane, which will bindto negatively charged molecules. Another preferred support is layeredsilicates, a group of laminated silica minerals that include, but arenot limited to: vermiculite, montmorillonite, bentonite, hectorite,fluorohectorite, hydroxyl hectorite, boron fluorophlogopite, hydroxylboron phlogopite, mica, and the like. Preferred micas are those that canbe fractured to produce a smooth surface, more preferably an atomicallysmooth surface.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, e.g., as laminates, to obtain various properties. For example,protein coatings, such as gelatin can be used to avoid non specificbinding, simplify covalent conjugation, enhance signal detection or thelike.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like.

D) Binding of the First Component to the Support

As indicated above, in preferred solid phase assays either the testagent and/or a cytoskeletal component is attached to a solid support.The manner of linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature (see, e.g.,Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York, 1978, andCuatrecasas, (1970) J. Biol. Chem. 245 3059). Adhesion of a cytoskeletalcomponent, and/or a test agent, to the solid support can be direct(i.e., the cytoskeletal component and/or test agent(s) directly contactthe solid support) or indirect via a linker (i.e., the linker is aparticular compound or compounds attached to the support, and thecytoskeletal component and/or test agent(s) bind to this compound orcompounds rather than to the solid support).

Method of attaching biological, and other, molecules to solid supportsare well known to those of skill in the art. For example, compounds havebeen immobilized either covalently (e.g., utilizing single reactivethiol groups of cysteine residues, Colliuod et al. (1993) BioconjugateChem. 4, 528-536)), or non-covalently but specifically (e.g., viaimmobilized antibodies (Schuhmann et al. (1991) Adv. Mater. 3: 388-391;Lu et al. (1995) Anal. Chem. 67: 83-87), the biotin/streptavidin system(Iwane et al. (1997) Biophys. Biochem. Res. Comm. 230: 76-80),metal-chelating Langmuir-Blodgett films (Ng et al. (1995) Langmuir 11:4048-4055; Schmitt et al. (1996) Angew. Chem. Int. Ed. Engl. 35:317-320; Frey et al. (1996) Proc. Natl. Acad. Sci. USA 93:4937-4941;Kubalek et al. (1994) J. Struct. Biol. 113:117-123) and metal-chelatingself-assembled monolayers (Sigal et al. (1996) Analytical Chem., 68:490-497) for binding of polyhistidine fusion proteins.

By manipulating the solid support and the mode of attachment of thecytoskeletal component to the support, it is possible to control theorientation of the cytoskeletal component. For example, copending patentapplication entitled “Reversible Immobilization of Arginine-taggedMoieties on a Silicate Surface, U.S. Ser. No. 60/057,929, filed on Sep.4, 1997, PCT/US98/18531 describes the use of an arginine tail to attachcytoskeletal proteins to a mica film.

Thus, for example, where it is desired to attach a myosin molecule to asurface in a manner that leaves the myosin tails free to interact withother molecules, a tag (e.g., polyarginine or polyglutamate, magneticparticle, etc.) may be added to the myosin molecule at a particularposition in the myosin sequence (for example, near the myosin head suchthat, when the myosin molecule is attached to a surface (e.g., asilicate surface by means of an arginine tag, an iron-containing surfaceby means of a magnetic tag, a metal (e.g. IMAC reagent) by means of ahistidine tag, etc.), the tail is free. One preferred site for theplacement of such an attachment tag is on loop two of the myosin head, aflexible external loop at the actin binding domain. Other sites includethe myosin carboxy terminus. Other cytoskeletal proteins, such askinesin, may similarly be modified to add an arginine tag to externalloops or carboxy or amino termini.

If polyglutamate is used, the tag minimally comprises 3-4, preferably6-8, and more preferably 10-15 glutamate residues. Proteins tagged witha polyglutamate tail will preferably bind to a positively chargedsurface, preferably aminosilane. Similar to the arginine tag, thepolyglutamate tag can be used to orient the tagged cytoskeletalcomponent, as described above. It is also possible to purifypolyglutamate-tagged molecules on regular anion exchange columns.

In one embodiment, the invention involves binding of polymer-interactingproteins to a surface coated with polymers. High adsorption of polymersis achieved by first shearing polymers to short sizes (e.g., about 1μ orless) and then adsorbing them onto surfaces coated with inactivatedmotor proteins that bind tightly to the polymer. A motor protein can beinactivated by mutating the “motor domain” such that hydrolysis ofchemical energy to produce mechanical force can no longer occur.Nonspecific binding is minimized by subsequent absorption of carrierprotein (e.g., bovine serum albumin).

In one embodiment, the polymer-interacting proteins are labeled (e.g.,fluorescently labeled), either chemically or by genetic fusion to afluorescent protein (e.g., GFP). The reverse assay is also possible inwhich the polymer-interacting protein is adsorbed onto the surface and afluorescently-labeled polymer is employed. If a binding interactionoccurs, fluorescent protein is depleted from the solution andaccumulates on the surface. A quantitative readout of fluorescence onthe surface is made, for example, by total internal reflection, whichselectively excites fluorescent molecules on the surface and not in thesolution.

E) Test Compositions

The materials and methods of this invention are particularly useful foranalyzing the effects of biological molecules on cytoskeletalinteractions. Compounds suitable of assay in the methods of thisinvention include, but are not limited to, proteins, glycoproteins,antibodies, saccharides, lipids, nucleotides, nucleotide analogues,nucleic acids (e.g., DNA, RNA, peptide nucleic acids, etc.), and organicmolecules, particularly small organic molecules.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents preferably comprise functional groups suitable forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In an embodiment provided herein, the candidate bioactive agents areproteins. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration. In the preferredembodiment, the amino acids are in the (S) or L-configuration. Ifnon-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations.

In another embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. In oneembodiment, the libraries are of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In one embodiment, the candidate agents are peptides of from about 5 toabout 30 amino acids, with from about 5 to about 20 amino acids beingpreferred, and from about 7 to about 15 being particularly preferred.The peptides may be digests of naturally occurring proteins as isoutlined above, random peptides, or random peptides. By “randomized” orgrammatical equivalents herein is meant that each nucleic acid andpeptide consists of essentially random nucleotides and amino acids,respectively. Since generally these random peptides (or nucleic acids,discussed below) are chemically synthesized, they may incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bioactiveproteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In another embodiment, the candidate agents are nucleic acids. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention is preferably single-stranded or doublestranded and will generally contain phosphodiester bonds, although insome cases, as outlined below, nucleic acid analogs are included thatmay have alternate backbones, comprising, for example, phosphoramide(Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein;Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J.Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487;Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am.Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; andU.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am.Chem. Soc. 111:2321, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm(1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed.Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996)Nature 380: 207). Other analog nucleic acids include those with positivebackbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097;non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423;Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al.(1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC SymposiumSeries 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic &Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, andChapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modificationsin Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev.pp 169-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of additional moietiessuch as labels, or to increase the stability and half-life of suchmolecules in physiological environments.

In addition, mixtures of naturally occurring nucleic acids and analogscan be made. Alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthine,hypoxanthine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidate agentsmay be naturally occurring nucleic acids, random nucleic acids, or“biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, the candidate agent is a small molecule. Thesmall molecule is preferably 4 kilodaltons (kd) or less. In anotherembodiment, the compound is less than 3 kd, 2 kd or 1 kd. In anotherembodiment the compound is less than 800 daltons (D), 500 D, 300 D or200 D. Alternatively, the small molecule is about 75 D to 100 D, oralternatively, 100 D to about 200 D.

As indicated above, the test composition(s) can be provided as membersof a “library” or “collection” of compounds. Such collections orlibraries can be produces simply by combining two or more different testcompositions. However for effective screening of a wide number of a widenumber of different test compositions preferred libraries contain alarge number of different compositions. Thus, library production oftenutilizes combinatorial chemical synthesis techniques to produce a“combinatorial chemical library”. A combinatorial chemical library is acollection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis by combining a number of chemical“building blocks” such as reagents. For example, a linear combinatorialchemical library such as a polypeptide library is formed by combining aset of chemical building blocks called amino acids in every possible wayfor a given compound length (i.e., the number of amino acids in apolypeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks (Gallop etal. (1994) 37(9): 1233-1250).

Such chemical libraries exist in a continuum between two functionalobjectives. “Broad screening” libraries are used to rapidly screen awide range of diverse agents. Thus “broad screening” libraries arecharacterized by large library size, broad structural diversity, nospecific structural goal and are typically synthesized utilizing a widenumber of different “building blocks.” At the other end of the spectrum,libraries are used for “chemical analoging” to provide subjects forscreening a wide variety of related chemical analogues. Chemicalanaloging libraries are typically of moderate library size, showrelatively narrow structural diversity, contain a relatively limitedrepertoire of building blocks are typically synthesized using a specificorder of combination of building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art (see, e.g., Gorden and Kerwin (1998)Combinatorial Chemistry and Molecular Diversity in Drug Discovery, JohnWiley & Sons, Inc. N.Y.). Such combinatorial chemical libraries include,but are not limited to, peptide libraries (see, e.g., U.S. Pat. No.5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghtonet al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means theonly approach envisioned and intended for use with the presentinvention. Other chemistries for generating chemical diversity librariescan also be used. Such chemistries include, but are not limited to:peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encodedpeptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al. (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a β-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer. Chem.Soc. 114: 9217-9218), analogous organic syntheses of small compoundlibraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658; Gordon etal., (1994) J. Med. Chem. 37: 1385), nucleic acid libraries (see, e.g.,Strategene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat.No. 5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996)Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines: Baum (1993) C&EN, January 18, page 33;isoprenoids: U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones: U.S. Pat. No. 5,549,974; pyrrolidines: U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds: U.S. Pat. No. 5,506,337;benzodiazepines: U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 NWS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, FosterCity, Calif.; 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate 11, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tfipos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

III. Antibodies, Antisera, and Immunoassays

In certain circumstances, it may be necessary to detect the presence andconcentration of certain compounds in solutions and in complex mixturesbefore performing the binding assays described above. One way of doingso is by the use of electrophoresis. A second way is to obtainpolyclonal and monoclonal antibodies using methods known to those ofskill in the art, and perform immunoassays (see, e.g., Coligan (1991),Current Protocols in Immunology, Wiley/Greene, NY; and Harlow and Lane(1989), Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Goding (1986), Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature,256:495-497).

Such techniques include antibody preparation by selection of antibodiesfrom libraries of recombinant antibodies in phage or similar vectors(see, Huse et al. (1989), Science, 246:1275-1281; and Ward et al.(1989), Nature, 341:544-546). For example, in order to produce antisera,a particular antigen a fragment thereof is isolated as described herein.For example, recombinant protein is produced in a transformed cell line.An inbred strain of mice or rabbits is immunized with the componentusing a standard adjuvant, such as Freund's adjuvant, and a standardimmunization protocol. Alternatively, a synthetic peptide derived fromthe antigen and conjugated to a carrier protein can be used animmunogen. Polyclonal sera are collected and titered against theimmunogen in an immunoassay. Polyclonal antisera with a titer of 10⁴ orgreater are selected and tested for their cross reactivity againstcytoskeletal components and test compositions or even other cytoskeletalcomponents and test compositions, using a competitive bindingimmunoassay. Specific monoclonal and polyclonal antibodies and antiserawill usually bind with a K_(D) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

A number of immunogens may be used to produce antibodies specificallyreactive with either cytoskeletal components and test compositions.Recombinant protein is the preferred immunogen for the production ofmonoclonal or polyclonal antibodies. Naturally occurring protein mayalso be used either in pure or impure form. Synthetic peptides madeusing the cytoskeletal components and test compositions sequencesdescribed herein may also used as an immunogen for the production ofantibodies to the protein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the cytoskeletal component.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purifiedcytoskeletal component, is mixed with an adjuvant and animals areimmunized. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the cytoskeletal components and test compositions. When appropriatelyhigh titers of antibody to the immunogen are obtained, blood iscollected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to thecytoskeletal component can be done if desired. (See Harlow and Lane,supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (See, Kohler and Milstein (1976) Eur. J. Immunol.6:511-519). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al. (1989) Science 246:1275-1281.

A particular antigen can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Basic and Clinical Immunology, 7th Edition (D. Stites andA. Terr ed.) 1991. Moreover, immunoassays of the present invention canbe performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, BocaRaton, Fla. (1980); “Practice and Theory of Enzyme Immunoassays,” P.Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publishers B.V. Amsterdam (1985); and, Harlow and Lane,Antibodies, A Laboratory Manual, supra.

Immunoassays to cytoskeletal components and test agents of the presentinvention may use a polyclonal antiserum raised against the cytoskeletalcomponent or test agent or a fragment thereof. This antiserum isselected to have low crossreactivity against other (non-cytoskeletalcomponents and test compositions or cytoskeletal components and testcompositions) proteins and any such crossreactivity is removed byimmunoabsorption prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the cytoskeletalcomponent is isolated as described herein. For example, recombinantprotein is produced in a transformed cell line. An inbred strain of micesuch as balb/c is immunized with the selected cytoskeletal componentusing a standard adjuvant, such as Freund's adjuvant, and a standardmouse immunization protocol. Alternatively, a synthetic peptide derivedfrom the sequences disclosed herein and conjugated to a carrier proteincan be used an immunogen. Polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Polyclonal antisera with a titer of 10⁴ or greater are selected andtested for their cross reactivity against non-cytoskeletal componentsand test compositions, using a competitive binding immunoassay such asthe one described in Harlow and Lane, supra, at pages 570-573.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the antigen can beimmobilized to a solid support. Proteins (other cytoskeletal componentsand test compositions, or non-cytoskeletal components and testcompositions) are added to the assay which compete with the binding ofthe antisera to the immobilized antigen. The ability of the aboveproteins to compete with the binding of the antisera to the immobilizedprotein is compared to the protein. The percent crossreactivity for theabove proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the proteinslisted above are selected and pooled. The cross-reacting antibodies areoptionally removed from the pooled antisera by immunoabsorption with theabove-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein, either cytoskeletal components and testcompositions. In order to make this comparison, the two proteins areeach assayed at a wide range of concentrations and the amount of eachprotein required to inhibit 50% of the binding of the antisera to theimmobilized protein is determined.

The presence of a desired polypeptide (including peptide, transcript, orenzymatic digestion product) in a sample may also be detected andquantified using Western blot analysis. The technique generallycomprises separating sample products by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with labelingantibodies that specifically bind to the analyte protein. The labelingantibodies specifically bind to analyte on the solid support. Theseantibodies are directly labeled, or alternatively are subsequentlydetected using labeling agents such as antibodies (e.g., labeled sheepanti-mouse antibodies where the antibody to an analyte is a murineantibody) that specifically bind to the labeling antibody.

IV. Data Management.

In one embodiment, the assays of this invention are facilitated by theuse of databases to record assay results. Particular with the use oflarge-scale screening systems, (e.g., screening of combinatoriallibraries) data management can become a significant issue. For example,all natural hexapeptides have been synthesized in a single combinatorialexperiment producing about 64 million different molecules. Maintenanceand management of even a small fraction of the information obtained byscreening such a library is aided by methods automated informationretrieval, e.g. a computer database.

Such a database is useful for a variety of functions, including, but notlimited to library registration, library or result display, libraryand/or result specification, documentation, and data retrieval andexploratory data analysis. The registration function of a databaseprovides recordation/registration of combinatorial mixtures and assayresults to protect proprietary information in a manner analogous to theregistration/protection of tangible proprietary substances. Library andassay result display functions provide an effective means to reviewand/or categorize relevant assay data. Where the assays utilize complexcombinatorial mixtures for test agents, the database is useful forlibrary specification/description. The database also providesdocumentation of assay results and the ability to rapidly retrieve,correlate (or statistical analysis), and evaluate assay data.

Thus, in some preferred embodiments, the assays of this inventionadditionally involve entering test agent(s) identified as positive(i.e., having an effect on cytoskeletal activity) in a database of“positive” compounds and more preferably in a database of therapeutic orbioagricultural lead compounds.

The database can be any medium convenient for recording and retrievinginformation generated by the assays of this invention. Such databasesinclude, but are not limited to manual recordation and indexing systems(e.g. file-card indexing systems). However, the databases are mostuseful when the data therein can be easily and rapidly retrieved andmanipulated (e.g. sorted, classified, analyzed, and/or otherwiseorganized). Thus, in a preferred embodiment, the signature the databasesof this invention are most preferably “automated”, e.g., electronic(e.g. computer-based) databases. The database can be present on anindividual “stand-alone” computer system, or a component of ordistributed across multiple “nodes” (processors) on a distributedcomputer systems. Computer systems for use in storage and manipulationof databases are well known to those of skill in the art and include,but are not limited to “personal computer systems”, mainframe systems,distributed nodes on an inter- or intra-net, data or databases stored inspecialized hardware (e.g. in microchips), and the like.

V. Kits.

In still another embodiment, this invention provides kits for practiceof the assay methods described herein. The kits preferably comprise oneor more containers containing one or more of the assay componentsdescribed herein. Such components include, but are not limited to one ormore cytoskeletal components, one or more test agent(s), solid supports(e.g., microtitre plates) with one or more attached components, buffers,labels, and other reagents as described herein.

The kits may optionally include instructional materials containingdirections (i.e., protocols) for carrying out any of the assaysdescribed herein. While the instructional materials typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Observation of Kinesin Motor Binding to a Surface-AdsorbedMicrotubule Detection by Total Internal Reflection Microscopy andModulation of that Binding by Nucleotide

To observe single motor molecule binding, motor-GFP fusions wereprepared. Previous studies had shown that single GFP molecules (theSer65Thr variant) can be detected by TIR microscopy (Pierce et al.,1997, Nature 388: 388). For the wild-type kinesin and the NK-1 chimera,GFP was fused C-terminal to residue 560. In the case of Ncd, GFP wasfused N-terminal to residue 236, which retains ¾th of the Ncdcoiled-coil stalk and the complete motor domain but lacks the N-terminaldomain that bundles microtubules (Chandra et al. (1993) J. Biol. Chem.268: 9005-9013.). This GFP fusion method is advantageous compared tofluorescent dye modification, which tends to inactivate the Ncdcatalytic domain. Hydrodynamic and single fluorescent spot intensityanalysis indicated that K560-GFP and Ncd-GFP are dimers under conditionsof the assay.

By using green fluorescent protein fused to the polymer-binding protein,this assay can be performed in crude cell extracts. GFP chimeras providetwo advantages. First, this alleviates the necessity of purifying thepolymer-interacting protein to homogeneity before performing the bindingassay. In many cases, purification and fluorescent labeling of manyproteins is too difficult, due to their limited quantities in cells.Second, the drug screening effort can be performed in the proteinenvironment of a whole cell extract. This is advantageous, since acomplex mixture of competing molecules for the drug is present.

A) Expression Constructs

A human conventional kinesin construct comprising residues 1-560 with aC terminal 6 histidine (6×His) tag was cloned into pET17B (Novagen,Inc.). The chimera NK-1 was constructed by performing PCR on the GST-MC1construct (Chandra et al., 1993, J. Biol. Chem. 268: 9005-9013) with the5′ oligo corresponding to a.a. 348-359 and the 3′ oligo corresponding toa.a. 656-667 to generate PCR product 1 (encoding a.a. 348-667). A secondPCR reaction was performed on the K560-6×His vector above using a 5′oligo corresponding to a.a. 323-333 and a 3′ oligo corresponding to a.a.7 and extending 14 b.p. into the vector promoter to generate a 3.9 kbproduct (PCR product 2) which included N- and C-terminal kinesinsequences as well as the intervening vector sequence. Products 1 and 2were treated with alkaline phosphatase and the Klenow polymerasefragment to ensure blunt ends. The two PCR products were then ligatedand transformed in DH5α cells. The C-terminal junction of the ligatedproduct had a base pair deletion which was later repaired by PCRmutagenesis. The repaired chimera was then subcloned between Nde1(a.a. 1) and Mun1 (a.a. 407) sites into the K560-6×His pET17B vector inorder to eliminate any PCR-induced errors in the vector. NK-2 wasgenerated by the same strategy as indicated above with the exceptionthat the 5′ oligo for PCR product I corresponded to a.a. 49-60 and the3′ oligonucleotide for PCR product 2 corresponded to a.a. 48-37. TheNK-3 construct was generated by Stratagene QuikChange protocol(Stratagene, Inc.) using oligonucleotides that substituted the Ncd L11sequence (a.a. 586-597) for the kinesin L11 sequence (a.a. 237-252) inthe K560-6×His pET17B vector. The remainder of the cloning was asdescribed in Woehlke et al. (1997), Cell 90: 207-216. All coding regionswere confirmed by DNA sequencing.

For GFP fusion proteins (see FIG. 1), PCR was used to introduce: 1) a 5′Nde1 site and a 3′ Kpn1 site at a.a. 560 of human ubiquitous kinesin andNK-1, and 2) a 5′ Kpn1 site and a 3′ 6×His tag plus an Xho1 site to GFPmutant S65T (from R. Tsien (UCSD)). K560 and NK-1 were then fused to GFPat the introduced Kpn1 site (which adds a Gly-Thr linker sequence) andcloned into pET17b between the Nde1 and Xho1 sites. To make the Ncd-GFPfusion, a 5′ Kpn1 site and a 3′ Xho1 site were added to Ncd a.a.236-700, and a 5′ Nhe1 site plus 6×His tag and 3′ Kpn1 site were addedto GFP (again adding a Gly-Thr linker sequence) via PCR. These productswere joined together into the Nhe1-Xho1 sites of pET17b. All PCR-derivedsequences were confirmed by DNA sequencing.

B) Bacterial Protein Expression

Constructs were transformed into E. coli strain BL21(DE3), grown inTPM-ampicillin (20 g/l tryptone, 15 g/l yeast extract, 8 g/l NaCl, 10 mMglucose, 2 g/l Na₂HPO₄, 1 g/l KH₂PO₄, and 100 μg/ml ampicillin) at 24°C. to an O.D.₆₀₀ of 1-2, and then protein expression was induced for9-14 hr with 0.2 mM IPTG. Cells were lysed by French press (0.8 MPa) in50 mM NaPO₄, 20 mM imidazole, 250 mM NaCl, 1 mM MgCl₂, 0.5 mM ATP, 10 mMP-mercaptoethanol (βME), leupeptin (1 μg/ml), pepstatin (1 μg/ml),chymostatin (1 μg/ml), aprotinin (1 μg/ml), and 0.25 mg/ml Pefabloc(Boehringer Mannheim)(50 ml buffer per 2 l culture). The supernatantfrom a 28,000×g, 30 min centrifugation was collected and incubated withNi—NTA resin (Qiagen, Inc.) for 1 hr at 4° C. (1-2.5 ml resin per 50 mlof supernatant). The mixture was then transferred to a disposablecolumn, and the resin was washed with 50 ml of 50 mM NaPO₄ (pH 6), 250mM NaCl, 1 mM MgCl₂, 0.1 mM ATP, and 10 mM βME. Proteins were elutedwith 50 mM NaPO₄, 500 mM imidazole-Cl, 250 NaCl, 1 mM MgCl₂, 0.1 mM ATP,and 10 mM βME (pH 7.2). The peak fractions were then diluted 5-fold intocolumn buffer supplemented with 50 mM NaCl and further purified bymono-Q chromatography. The K560-GFP and NK-1-GFP fusion proteins werepurified by mono-Q chromatography with elution at 0.35 M NaCl in a 16 ml0.2-1.0 M gradient in 25 mM NaPipes (pH 6.8; K560-GFP) or 10 mM NaPO₄(pH 7.2; NK-1-GFP) with 2 mM MgCl₂, 1 mM EGTA, 1 mM DTT, and 0.1 mM ATP.Ncd-GFP was further purified by mono-S chromatography with elution at0.3 M NaCl in a 30 ml 0.1-1.1 mM NaCl gradient in 10 mM NaPO₄ (pH 7-2),2 mM MgCl₂, 1 mM EGTA, 1 mM DTT, and 0.1 mM ATP. The motor-GFP fusionproteins were then subjected to an additional microtubule affinitypurification step by incubating with microtubules and 1 mM AMPPNP,centrifuging the motor-microtubule complex and releasing the activemotor from the microtubule with 5 mM MgATP/200 mM KCl.

For all preparations, 10-20% sucrose was added to peak fractions beforefreezing and storage in liquid nitrogen. Protein concentrations werecalculated by running the kinesin along with a BSA-standard curve on aSDS polyacrylamide gel, staining with Coomassie, capturing the gel imagewith a ccd camera, and then measuring optical densities using thecomputer program NIH Image.

Motors from the peak mono-Q fractions were adsorbed onto glass surfacesof microscope flow cells at concentrations of 0.5-10 μM. For motor-GFPfusion proteins, affinity-purified anti-GFP polyclonal antibodies (0.5mg/ml) were first adsorbed onto the glass surface, the flow cell waswashed with buffer, and the motors were then allowed to bind to theantibody-coated surface. A buffer containing 15 mM NaMOPS (pH 7), 50 mMNaCl, 20 μM taxol, 10 μg/ml rhodamine-labeled microtubules (Hyman etal., 1990, Meth. Enzym. 196: 303-319.), 1 mM ATP, 1 mM EGTA, 2 mM MgCl₂,1 mM DTT, 2 mg/ml casein, and an oxygen depletion system composed of 22mM glucose, 0.5% P-mercaptoethanol, 0.2 mg/ml glucose oxidase, and 36μg/ml catalase (Harada et al., 1990, J. Mol. Biol. 216: 49-68.). Bindingwas also observed in the lower ionic strength buffers employed in thesingle molecule fluorescence motility assays.

Polarity marked microtubules (Hyman (1991) J. Cell Sci. Supp. 14:125-127) were prepared by first polymerizing short microtubules fromrhodamine-tubulin and 0.5 mM GMPCPP (a nonhydrolyzable GTP analog)—Thesebrightly labeled microtubules were then used as seeds to polymerize amore dimly labeled microtubule segment with 0.1 mg/ml rhodamine-labeledtubulin, 1.5 mg/ml unlabeled tubulin, and 1.5 mg/ml NEM-modified tubulin(Hyman et al., 1990, Meth. Enzym. 196: 303-319.) which inhibitsminus-end growth. Polarity marked microtubules were used.

For the single molecule fluorescence assay, motor-GFP proteins werediluted to a concentration of 1-50 nM in a buffer containing 1 mM ATP, 1mM EGTA, 2 mM MgCl₂, 7.5 mg/ml bovine serum albumin (BSA) as carrierprotein, and the oxygen depletion system described above. Kinesin-GFPwas standardly assayed in the above solution with 12 mM KPipes (pH 6.8).A variety of buffer conditions were also used, including 12 mM KPipes(pH 6.8), 12 mM KMOPS (pH 7), 50 mM KMOPS (pH 7), and 50 mM KMOPS (pH 7)with 50 mM NaCl.

C) Microscopy and Analysis

Rhodamine-labeled microtubules were illuminated with a 100 W mercurylamp and imaged by epifluorescence microscopy using a 60×, 1.4 N.A.objective (Olympus, Inc). The image was projected onto asilicon-intensified target camera (Hamamatsu, Inc.) and then recordedonto SVHS tape.

For single molecule fluorescence imaging, 4 μl of assay mix describedabove was spotted onto a cleaned quartz slide, covered with an 18 mmcoverslip, sealed with rubber cement, and imaged on a low-background TIRoptical bench microscope constructed by the authors (Pierce and Vale,1997). Briefly, an argon-ion laser was used at 488 nm and 5 mW to exciteGFP, and a HeNe laser was operated at 0.4 mW to excite sea urchinaxonemes (prepared as described by Gibbons and Fronk (1979), J. Biol.Chem. 254: 187-196) and labeled with Cy5 dye (Vale et al., 1996, Nature380, 451-453). Laser illumination was passed through a X/4 plate set toproduce circularly polarized light and focused by 25 cm lens at anappropriate angle through a prism to produce total internal reflectionand evanescent field illumination of a ˜30×40 mm area at the sample(Funatsu et al., 1995, Nature 374: 555-559). Fluorescence was collectedby a Nikon PlanApo 100/1.4 objective, collimated, passed through customdesigned dichroic mirror and barrier filters and focused onto a CCDcamera coupled to a selected SR UB Gen3+ intensifier tube from StanfordPhotonics Inc. Data was recorded to video tape after contrastenhancement by an Argus-20 image processor (Hamamatsu Photonics, Inc.).Behavior of single GFP molecule fluorescence is described elsewhere(Pierce et al., 1997, Nature 388: 388).

When K560-GFP was combined with axonemal microtubules and ATP,individual fluorescent spots could be easily observed binding to theaxoneme, as shown previously (Pierce et al., 1997, Nature 388: 388). Incontrast, at 10 nM NK-1-GFP and Ncd-GFP, fluorescent spots did notassociate with axonemes at more than background levels, indicating thatmicrotubule associations must be very transient. The capacity of Ncd-GFPand NK-1-GFP to bind to microtubules in this assay was demonstrated byinducing a strong microtubule binding state by depleting ATP, whichresulted in the association of the majority of fluorescent moleculeswith the axoneme

The above method involves selective surface illumination and viewingwithout removing free protein in solution. However, using an extra stepof physically removing the solution, a fluorescence reading of thesurface can be made using a conventional fluorimeter.

Example 2 Reversible, Site-Specific Immobilization of Arginine-TaggedFusion Proteins on Mica Surfaces

This example describes the specific binding of polyarginine taggedproteins to atomically flat negatively charged mica surfaces. Thepolyarginine tags were expressed as fusion proteins. It is shown hereinthat the arginine (e.g., hexaarginine) tagged proteins bind to mica viathe Arg-tag based on ion exchange of naturally occurring potassiumcations. Only nonspecific binding was observed with the control proteinthat is free of the Arg-tag. This novel technology facilitates theuniform and specific orientation of immobilized proteins on a standardsubstrate used for many surface-related applications.

A) Materials and Methods

Muscovite mica was obtained from Provac (Liechtenstein). The plasmidpGFPuv was from Clontech (Palo Alto, Calif.) and the vector pET28a(+)was from Novagen (Madison, Wis.). All other reagents were from SigmaChemical (St. Louis, Mo.) and of highest available grade. Ultrapurewater with a resistance of 18 Nffcm was used for all aqueous buffers(purified by passage through a Mlli-Q purification system).

B) Preparation of GFPH6, GFPH6R6, GFPR6

For the addition of six histidine residues to the N-terminus of GFP, twooligodeoxyribonucleotide primers were designed: one corresponding to theN-terminal part of the GFP gene (5′-GGA ATT CCA TAT GAG TAA AGG AGA AGAACT TTT C-3′, designated primer #1, SEQ ID No: 1) and a secondcorresponding to the C-terminal part (5′-GAC CGG CGC TCA GTT GGA ATTC-3′, designated primer #2, SEQ ID No: 2). Theseoligodeoxyribonucleotides were used for PCR with 20 ng of linearizedpGFPuv as template. The amplified fragments, digested with Nde1 andBamH1, were ligated with the linearized expression vector pET28a(+). Theresulting plasmid pGFPH6 was used for transformation of E. coliBL21(DE3). Standard protocols were followed for DNA handling andbacterial transformation (Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor: Cold Spring Harbor LaboratoryPress).

To introduce a tag of six arginine residues on either the N- orC-terminal part of GFP, the same procedure was used with the followingoligodeoxyribonucleotides: (primer #2) and 5′-GGA ATT CCA TAT GCG CCGTCG CCG TCG CCG TAT GAG TAA AGG AGA AGA ACT TTT C-3′ for GFPH6R6,(primer #1) and 5′-TTG GAA TTC ATT AGC GAC GGC GAC GGC GAC GCG CGG TGCCTT TGT AGA GCT CAT CCA TG-3′ for GFPR6. The PCR and cloning procedurewas performed as described above. The resulting plasmids pGFPH6R6 andpGFPR6 were used to transform E. coli BL21(DE3).

C) Expression and Purification of the Recombinant Proteins

All of the expressed proteins carry a vector-encoded tag of ahexa-histidine sequence for purification by metal chelate affinitychromatography on a Ni²⁺/NTA matrix (Qiagen, Santa Clarita, Calif.). Thecells were grown at 37° C. by shaking in LB-medium containing 25 mg/mlKanamycin. At an OD₆₀₀ of 0.8 the cells were induced with 1 mM IPTG, and5 h later, they were harvested by centrifugation at 6000×g for 10 min.The cells were lysed by addition of lysozyme at a concentration of 100mg/ml and 10% (v/v) of 1% Triton X-100 in 50 mM Tris-HCl pH 7.5, 50 mMKCl, 1 mM EDTA. After incubation for 30 min on ice, MgCl₂ was added to afinal concentration of 40 mM. The liberated DNA was digested by adding0.2 mg DNaseI per ml lysate. The lysate was incubated for 15 min on iceand then centrifuged at 30,000×g for 40 min. The clear supernatant wasdialyzed against buffer containing 10 mM Hepes/NaOH pH 7.4, 50 mM NaCl,and then applied to a Ni²⁺/NTA column. Weakly bound proteins were elutedwith 10 mM imidazole pH 8.0. The his-tagged proteins were eluted with500 mM imidazole in the case of the GFPH6 and with 500 mM imidazole, 500mM NaCl for all the other variants (the Arg-tag caused a strong ionicinteraction with the Ni²⁺/NTA matrix). The eluted proteins were dialyzedagainst buffer containing 10 mM Hepes/NaOH pH 7.4, 50 mM NaCl, 50%glycerol and stored at −20° C. The purity of the recombinant proteinswas estimated by SDS-polyacrylamide gel electrophoresis and found to begreater than 95%.

D) Protein Adsorption to Mica

Mica sheets were cut into pieces of 5×5 cm and freshly cleavedimmediately before use. Droplets of protein solutions (GFPH6, GFPR6,GFPH6R6) at a concentration of 10 mg/ml were applied onto the previouslyunexposed, hydrophilic surfaces resulting in 2 aqueous films ofapproximately 4 cm² in size. After incubation for 5 min, the mica sheetswere washed with 10 ml of water. The central parts, 1 cm² in size, werethen cut out to ensure that no contaminants from the edges could falsifythe subsequent analyses. For each data point four surfaces were analyzedand the readings were averaged. These surfaces, stored separately inEppendorf tubes, were then subjected to consecutive one-min washingsteps with 400 ml 10 mM Hepes/NaOH buffer pH 7.4 containing increasingconcentrations of salt with different mono- and bivalent cations (50,125, 250 mM, Na⁺, K⁺, Mg²⁺). For quantitation of active, adsorbed GFP,the eluates were collected separately and analyzed by fluorescencemeasurement at 509 nm (excitation at 395 nm) using an SLM8000spectrophotometer (Aminco, Silver Spring, Md.) and GFP of knownconcentration as standard.

Qualitative determination of immobilized GFP was carried out with X-rayphotoelectron spectroscopy (XPS) using the N1s narrow scans normalizedagainst the corresponding Si2s peaks, an element that does not occur inproteins. For this purpose the adsorbed proteins were washed with thesame salt-containing solutions as mentioned above (without buffer) andfinally rinsed with ultrapure water and dried under a stream ofnitrogen. This ensured that the XPS spectra were not dominated bycrystallized salts.

D) X-Ray Photoelectron Spectroscopy (XPS)

XPS was carried out on a Surface Science Model 150 XPS spectrometer withan AlKα source (1486 eV), a quartz monochromator, hemisphericalanalyzer, and a multichannel detector. A nickel grid, directlypositioned above the samples, and a charge neutralizer were used toprevent artifacts due to charging effects. The spectra were accumulatedat a take-off angle of 35″ and an angular acceptance of 30″, with a250×1000 μm spot size at a pressure of less than 1×10⁻⁸ Torr. The N1speaks shown in this example were normalized against Si2s and correctedfor the number of scans and the atomic sensitivity factors.

The results show that only about 1 pmol of the Arg-tag free GFPH6remained bound after extensive washing with 10 mM Hepes/NaOH, pH 7.4. Incontrast about 3 pmol of Arg-tagged GFP remained bound after this wash.Essentially all of the Arg-tag free GFPH6 was removed from the surfaceby consecutive washing steps with increasing concentrations of NaCl. Incontrast, only about 50% of the two GFP variants comprisinghexaarginine-tags (GFPR6H6 and GFPR6) came off with NaCl. The completerelease could be achieved by elution with arginine-containing washbuffer. It is likely that this arginine-releasable protein wasexclusively bound via its Arg-tag, whereas that released in the NaClwashing steps stemmed primarily from protein electrostatically bound tothe surface via other charged groups in the protein.

Example 3 Solution-Phase Assays: Use of ATPase Assays in ScreeningMicrotubule Binding Agents

A) Preparation of Sponge Extract for Screening.

The sponge Adocia (Haliclona) sp. (Collection #95-100) was collected inPalau, Western Caroline Islands, and was quickly frozen. The frozensponge (225 g) was diced and steeped in a mixture of dichloromethane(300 mL) and methanol (1 L) for 24 h. The solids were removed byfiltration and the solution was reduced in volume to 300 mL andextracted with dichloromethane (2×200 mL).

The aqueous phase was lyophilized to yield a pale yellow powder. Thepowder (1.0 g) was chromatographed twice on a reversed phase C18Sep-Pak, using a gradient of 30% MeOH in H₂O to 100% methanol (MeOH) aseluant, to obtain pure fractions containing adociasulfate-1 andadociasulfate-2 and a mixed fraction containing adociasulfates. Themixed fraction was separated by reversed phase HPLC using 1:1 MeOH—H₂Oas eluant. Pure fractions were combined to obtain adociasulfate-1 (13.5mg), adociasulfate-2 (14.1 mg) and adociasulfate-3 (3.3 mg).

B) Motility Assay.

TI-γ (a kinesin superfamily member from the fungus Thermomyceslanuginosus) was adsorbed to a glass coverslip and supplemented with amixture of microtubules, 2 mM Mg-ATP, and sponge extracts in DMSO (5%final concentration). Motility was scored visually on a Zeiss Axioplanmicroscope sat up for DIC and fitted with an Argus 10 video processor(Hamamatsu).

C) Proteins.

All kinetic and binding measurements were performed on a bacteriallyexpressed Drosophila kinesin heavy chain fragment containing amino acids5-351 of the wild type protein and a hexahistidine tag at theC-terminus. Protein was purified from the soluble fraction of IPTGinduced bacterial cells by a single round of affinity chromatography onNi—NTA-agarose (Qiagen), concentrated by microfiltration, and frozen insmall aliquots in liquid nitrogen.

D) Steady State Kinetics.

Initial rate measurements were done at room temperature using amalachite green assay (Geladopoulos et al. (1991) Anal. Biochem., 192:112-116) modified to work in 96 well microtiter plates and scored on aplate reader at 650 nm. ATP concentration dependence and basal ATPaserate were determined by a coupled enzymatic assay with pyruvate kinaseand lactate dehydrogenase monitoring changes in absorbance at 340 nm.Phosphate standards (650 μM-7 μM) were included with each reading.

E) ATPase Assay (ADP Release).

The percent of ADP released from the enzyme was determined by themethods of Hackney (see, e.g., Hackney (1994) J. Biol. Chem., 2690:16508-16511). Briefly, 80 μM kinesin was preincubated with α-³²PATP atroom temperature for 15 min and than stored on ice. 1 μl aliquots ofthat mixture were diluted into 100 μl of “chase mix” containing 0.5mg/ml pyruvate kinase, 2 mM phosphoenalpyruvate, and varyingconcentrations of adociasulfate. At different time points 5 μl aliquotsof the chase mix were quenched in 100 μl 1 M HCV/1 mM ATP/1 mM ADP. Theamount of ADP that became accessible to pyruvate kinase and wasconverted to ATP was determined by a thin layer chromatography onPEI-cellulose followed by phosphoimager quantitation.

F) Results

Extracts from 268 marine sponges were initially tested for their abilityto disrupt normal behavior of microtubules in a gliding motility assay.This screening method allowed immediate distinction between substancesthat affected microtubule movement and those that caused microtubuledepolymerization or breakage. Active extracts from the initial screeningwere then tested for inhibition of the microtubule-stimulated kinesinATPase.

The most promising candidates were extracts from the sponge Adocia sp.In the motility assay, these extracts disrupted microtubule attachmentto the kinesin-coated surface, and totally abolished movement. Themicrotubule stimulated ATPase of kinesin was also completely inhibited.

Three active compounds in the extract were identified and isolated.These specific compounds are referred to herein as adociasulfates whilethe generic compounds are referred to as Adocia compounds or Adociakinesin inhibitors. The structure of the Adocia compounds oradociasulfates does not resemble that of nucleotide triphosphates. Thisindicates that the Adocia structures are different from known kinesininhibitors. In addition, it is believed that the activity spectrum ofthe Adocia compounds is narrower than that of nucleotide triphosphatesor analogues thereof.

To further investigate specificity, one adociasulfate was tested on avariety of ATPases using the ATPase activity assay described above. Ofthose tested, the only enzymes substantially inhibited by adociasulfateare members of kinesin superfamily (Table 2).

TABLE 2 Concentrations of adociasulfate causing 50% inhibition ofenzymatic activity. enzyme C50 rabbit kidney ATPase >136 μM*Apyrase >136 μM* Myosin II (EDTA)^(A) 75 μM CENP-E 10 μM^(D) K5-351^(B)2 μM^(E) K411^(C) 2 μM^(E) TI-γ 2 μM^(E) ncd — myosin — pyruvate kinase— *enzyme was not inhibited by 50% at the highest used inhibitorconcentration of 136 μM.; ^(A)EDTA activated ATPase; ^(B)constructcontaining amino acids 5–351 of Drosophila kinesin; ^(C)constructcontaining first 411 amino acids of Drosophila kinesin; ^(D)at 6 μMtubulin; ^(E)at 2 μM tubulin

The behavior observed in the motility assay indicated thatadociasulfates interfere with microtubule binding to the motor. This wastested by performing a kinesin-microtubule co-sedimentation assay in thepresence of a nonhydrolysable ATP analog, AMP-PNP, with or withoutadociasulfate. Addition of adociasulfate abolished binding of kinesin tomicrotubules under these conditions.

Consideration of the kinesin mechanochemlcal cycle suggests that theeffect on microtubule binding could be induced either by looking thekinesin in a weakly-binding state resembling the kinesin-ADPintermediate by adociasulfate binding in the nucleotide pocket, or bydirect interference with the microtubule-binding site. Steady statekinetic measurements demonstrated that the adociasulfate-inducedinhibition is competitive with microtubules, and could be totallyreversed by high microtubule concentrations.

In contrast, varying the ATP concentration had no effect on the overallshape of the kinetic curves. V_(max) was progressively lower at higheradociasulfate concentrations. An additional argument againstadociasulfate binding at the nucleotide pocket comes from the lack of aninhibitory effect on the basal, non microtubule-stimulated rate of thekinesin ATPases. If adociasulfate interfered with nucleotide binding, orlocked the enzyme in a particular nucleotide-bound state, ATP turnoverin the absence of microtubule should be decreased. However,concentrations of up to 136 μM adociasulfate (the highest tested) didnot inhibit the basal ATPase rate.

Microtubule binding to kinesin induced 1,000-fold stimulation of thebasal ATPase rate, owing primarily to accelerated ADP release. It wastested whether adociasulfate binding to kinesin could mimic the effectof the microtubule by examining ADP release from kinesin in the presenceof varying concentrations of adociasulfate. Indeed, bursts of ADPrelease were observed and their magnitude correlated positively with theconcentration of adociasulfate. The adociasulfate concentration at 50%of maximum burst is much higher than the K₁ determined in steady statemicrotubule competition assays. This discrepancy may reflect differentaffinities for adociasulfate in different nucleotide states of kinesin.Steady state kinetic measurements of K₁ reflect the affinity of the mosttightly bound state of the entire cycle, which includes severalkinesin-nucleotide intermediates (K-ATP, K-ADP-Pi, K-ADP etc.).

In contrast, the ADP release experiment with adociasulfate involved onlyone state, K-ADP. It is intriguing that this state also has the lowestaffinity for the microtubule. It was initially surprising thatadociasulfate did not stimulate the kinesin basal ATPase even though itinduced ADP release. However, during steady state kinetic measurements,each single headed kinesin molecule must undergo several cycles ofattachment-detachment to microtubule subunits. In contrast, ASpresumably remains bound through multiple enzymatic turnovers. Thephysiological equivalent of such a state would be a kinesin moleculepermanently attached to a single tubulin dimer, a state for which nokinetic data exist. However, if the adociasulfate binding to kinesinresembles microtubule binding, the initial association event shouldresult in a burst of ADP release as observed.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. Allpublications, patents and patent applications mentioned in thisspecification are hereby incorporated by reference in their entirety forall purposes, to the same extent as if each individual publication,patent or patent application had been specifically and individuallyindicated to be incorporated by reference.

1. A method of identifying a therapeutic lead compound that modulatesactivity of a cytoskeletal system, said method comprising: i) providingan assay mixture comprising a first component of said cytoskeletalsystem and a second component of said cytoskeletal system, wherein saidcytoskeletal system is a microtubule system, and wherein said firstcomponent comprises a kinesin motor protein and said second componentcomprises a tubulin protein that specifically bind to each other; ii)contacting said assay mixture with a test compound to be screened forthe ability to inhibit or enhance binding between said first componentand said second component; iii) detecting a change in coupling betweenATP hydrolysis and force generation; wherein said change indicates thatsaid test compound modulates activity said cytoskeletal system.
 2. Themethod of claim 1, further comprising step iv) testing said testcompound on a variety of ATPases.
 3. The method of claim 1, wherein saidassay mixture comprises a cell lysate.
 4. The method of claim 1, whereinat least 50 test compounds are screened.
 5. The method of claim 2,wherein said variety of ATPases comprises CENP-E.
 6. The method of claim2, wherein said variety of ATPases comprises kinesin.
 7. The method ofclaim 2, wherein said variety of ATPases comprises TI-gamma.